Hybridization by an Electrical Force and Electrochemical Genome Detection Bull. Korean Chem. Soc. 2005, Vol. 26, No. 3
379
Articles
Hybridization by an Electrical Force and Electrochemical Genome Detection
Using an Indicator-free DNA on a Microelectrode-array DNA Chip
Yong-Sung Choi,* Kyung-Sup Lee,† and Dae-Hee Park
School of Electrical, Electronics and Information of Engineering, WonKwang University,
344-2 ShinYoung-Dong, IkSan 570-749, Korea. *E-mail: [email protected]
†
Dept. of Electrical Eng., Dongshin University, 252 DaeHo-Dong, Naju 520-714, Korea
Received August 31, 2004
This research aims to develop DNA chip array without an indicator. We fabricated microelectrode array by
photolithography technology. Several DNA probes were immobilized on an electrode. Then, indicator-free
target DNA was hybridized by an electrical force and measured electrochemically. Cyclic-voltammograms
(CVs) showed a difference between DNA probe and mismatched DNA in an anodic peak. Immobilization of
probe DNA and hybridization of target DNA could be confirmed by fluorescent. This indicator-free DNA chip
microarray resulted in the sequence-specific detection of the target DNA quantitatively ranging from 10−18 M
to 10−5 M in the buffer solution. This indicator-free DNA chip resulted in a sequence-specific detection of the
target DNA.
Key Words : Indicator-free DNA chip, Immobilization, Target DNA, Hybridization, Ferricyanide
Introduction
The development of DNA chips, miniaturized arrays of
immobilized single-strand DNA, is motivated by their
potential applications in disease diagnosis and genome
sequencing.1-4 In an array-based sensor, DNA probes of
varying sequence are immobilized on a surface where the
location of each probe is known. DNA diagnostics are
performed by exposing the array to a solution containing
single-strand DNA of unknown sequence, the target, that
can be labeled with a fluorescent 5 or a radioactive marker.
The sequence of the target is determined by imaging the
array and correlating the position of “hot spots” with a probe
sequence.
DNA-based sensors have potential applications that range
from genomic sequencing to mutation detection and pathogen identification.1-4,6 Conventional DNA chip systems
employ confocal fluorescence detection.7-10 Fluorescent
detection DNA chips and microarray scanners are too
expensive to use. Electrochemical detection is superior to
fluorescent detection as regards the cost, portability and
convenience. Recently, electrochemical DNA sensors11-15
have been developed using electrochemically active intercalators. The above mentioned researches have been
performed using fluorescent substances or indicators.
Thorp16,17 used Ru (bpy)32+ (bpy=2,2'-bipyridine) as a
detection marker for hybridization reaction and detected a
single base pair mismatch.
Steel4 and Hahn18 et al. have reported electrochemical
quantization of DNA using ferricyanide. However, no
integrated multichannel electrochemical DNA sensor has
been developed. Microfabrication technology has already
been established and enables to advance miniaturization and
mass production of DNA chips. An integrated multichannel
DNA chip can be microarray and this has advantages of lowcost and high through-put.
Therefore, this research aims to develop the multi-channel
label-free DNA chip array that has the above advantages to
solve the problems. At first, we fabricated a DNA microarray by microfabrication technology. It is able to detect
various genes electrochemically after immobilization of
various probe DNAs and hybridization of label-free target
DNA on the electrodes simultaneously. Probe DNAs
consisting of thiol group at their 5’-end were immobilized
on the gold electrodes. Then indicator-free target DNA was
hybridized by an electrical force and measured electrochemically. Redox peak of cyclic-voltammogram showed a
difference between target DNA and mismatched DNA in an
anodic peak current. This indicator-free DNA chip microarray resulted in the sequence-specific detection of the target
DNA quantitatively ranging from 10−18 M to 10−5 M in the
buffer solution. It suggested that this DNA chip could
recognize the sequence specific genes.
Experimental Section
Materials and Instrumentation. SH-p72 probe DNA
(5’-HS-AGGCTGCTCCCCCCGTGGCC-3’; MW: 6207.3,
Tm: 80.5 oC), mismatched DNAs, SH-m72 (5’-HS-AAGCTGCTCCCCCCGTGGCC-3’; MW: 6191.3, Tm: 78.5 oC)
and SH-R72 (5’-HS-AGGCTGCTCCCCGCGTGGCC-3’;
MW: 6247.3, Tm: 80.5 oC), having a thiol group19,20 at their
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Bull. Korean Chem. Soc. 2005, Vol. 26, No. 3
Figure 1. Fabrication process of microelectrode array for DNA
chip.
5’-end and these target DNA [p72; 3’-TCCGACGAGGGG GGCACCGG-SH-5’], which was complementary to
the probe, were synthesized and purified. The thiol modified
DNA probes were purified to remove a protection group
with NAP 10 columns (Pharmacia). The concentrated stock
solutions were stored at 5 oC in 10 mM Tris-HCl (pH 8.0)-1
mM EDTA solutions denoted as TE buffers. Micropipette
was used to immobilize probe DNAs.
Potassium ferricyanide (K3Fe(CN)6) and potassium
chloride (KCl) were purchased from Wako Pure Chemicals,
Ltd. (Tokyo, Japan). Other reagents were commercially
available or were laboratory grade. High-purity water (=18.2
MΩ·cm) was used in all the solutions.
The electrochemical measurements were carried out using
an electrochemical analyzer manufactured by CH Instruments Inc. Systems (Model 1030) and a computer system
with data storage. Voltammetric experiments were carried
out at 25 ± 1 oC in a conventional cell with three electrodes
[a reference electrode Ag/AgCl (Bioanalytical Systems), a
counter electrode Pt and another electrode mentioned later].
Unless otherwise indicated, voltammetry was carried out at
100 mV/s in 5 mM potassium ferricyanide and 100 mM
potassium chloride solution. Cyclic-voltammetry (CV) was
measured in the range of −100 ~ +700 mV at 100 mV/s. CV
took the data of the 5th cycles of potential scans.
Fabrication of Microelectrode Array. Figure 1 shows
fabrication process of micro-electrode array. About 200 nm
gold layer was deposited over a 20 nm titanium adhesion
layer on a glass by RF sputtering. Next, the chip was spincoated with positive photoresist (S1818) and baked at 110 oC
for 1 min in a oven (CLEAN OVEN PUHC-211, ESPEC).
UV light (MA-10, MIKASA) was irradiated to a resist film
for 25 sec through a photo-mask. It was developed to form
electrode, lead wires, and their connections by dipping it in
developer (MF319) and filtrated water for 60 sec. The lead
wires were photolithographically covered with photoresist
for insulation. Eight individually addressable gold electrodes
(diameter: 0.7 mm) and a Pt counter electrode were arranged
on the chip. Each microelectrode was connected to an
external potentiostat by insulated gold track.
Immobilization of Probe DNA. The gold electrodes were
reversibly cycled in a 10 mM H2SO4 solution from 0 to 1.7
V (vs Ag/AgCl) at 100 mV/s until an ideal redox wave of
Yong-Sung Choi et al.
H2SO4 was observed. Then, the electrodes were immersed in
the solution of the DNA probe and the mismatched DNAs (5
µM, 1 µL) for 2 h at 25 oC, and allowed to react utilizing the
affinity between gold and thiol group and washed with
distilled water to remove probes which were not adsorbed.
Electrochemical Gene Detection with the DNA Chip
Microarray. The electrode modified with double-stranded
20-mer DNA (dsDNA-electrode) was prepared by the
following procedure. An electric field was applied to the
electrode modified with the single strand DNA probe
(ssDNA-electrode) to hybridize the targeted gene (1 aM-50
µM, 1 µL), which was complementary to the probe as the
target DNA, at 300 mV for 5 sec using a probe station. After
the reaction, the electrodes were sequentially treated with a
reverse voltage and washed with the purified water for 1 min
at room temperature to remove the non-specifically bound
DNAs. The specific hybrids (dsDNA) were formed on the
ssDNA-electrode.
After washing the electrodes, the immobilized DNA probe
on the gold electrode was confirmed by voltammetric
method using 5 mM potassium ferricyanide in 100 mM
potassium chloride solution at 100 mV/s. The modified
electrodes were stored in TE buffers. The DNA prevents a
redox response of potassium ferricyanide ion after immobilization or hybridization on the Au surface.
Results and Discussion
Fabrication of Microelectrode Array. Figure 2 shows
microelectrode chip array including 8 gold electrodes and a
Pt counter electrode. Each gold electrode was formed with
electrode, lead wire, and their connection. The lead wire was
covered with photoresist for insulation. Eight individually
addressable gold electrodes and a Pt counter electrode were
arranged on the chip. Each microelectrode was connected to
an external potentiostat by insulated gold track.
Confirmation of Immobilization of Probe DNA and
Hybridization of Target DNA. Figure 3 shows the results
that confirmed immobilization of the probe DNA (SH-p72,
5 µM) and hybridization of the target DNA (p-72, 1 µM).
The target DNA was modified with FITC (fluorescence
Figure 2. Photograph of the DNA chip microarray with 8 channels
and 1 counter electrode.
Hybridization by an Electrical Force and Electrochemical Genome Detection Bull. Korean Chem. Soc. 2005, Vol. 26, No. 3
Figure 3. Confirmation of immobilization and hybridization. (a)
Immobilization of the probe DNA modified with SH and FITC. (b)
Hybridization of the target DNA modified with FITC by an electric
force. (c) No SH group was modified to the probe DNA. (d) The
mismatched target DNA was hybridized to the probe DNA by an
electric force.
isothiocyanate) and detected using a fluorescence microscope.
In Figure 3 (a) and (b), because a fluorescent was confirmed, we could know that probe DNA was immobilized and
the target DNA was hybridized to Au electrode.
However, we were able to know that the probe DNA was
immobilized with a SH group if a SH group was not
modified because the fluorescence was not observed (Figure
3(c)). On the other hand a fluorescent was not observed if
probe DNA was hybridized with mismatched target DNA
(Figure 3(d)).
Immobilization of Probe DNA. The DNA probe having
the thiol group was immobilized on the gold electrode.
Cyclic-voltammograms of potassium ferricyanide with the
bare gold electrode and the electrode modified with the
DNA probe (ssDNA-modified, 5 µM) are shown in Figure
4. In Figure 4(a) and (b), the peak currents of potassium
ferricyanide decreased about 59.5% and the peak-to-peak
separation (Ep) increased when the ssDNA-electrode was
used compared with that of the bare electrode (Figure 4(a)).
On the other hand, the gold electrode treated with the DNA
probe without the thiol group made no changes in the current
and Ep (data not shown). This suggested that the DNA on the
electrode blocked the electrochemical reaction between
DNA and the anionic redox couple ions. This result shows
that the DNA probe is immobilized on the gold electrode
through the thiol group of the 5’-end. The ssDNA-electrode
was used for the detection of a specific gene.
The ssDNA (SH-p72, 5 µM) was used for specific gene
detection. When biological molecules are exposed to the
electric field, molecules with a negative charge move rapidly
to an area with a positive charge. Thus, hybridization of
DNA having the negative charge through electronics is more
accelerated than traditional passive methods. The ssDNA-
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Figure 4. Cyclic-voltammograms of 5 mM potassium ferricyanide
in 100 mM potassium chloride at 100 mV/s using (a) bare gold
electrode, (b) probe-modified electrode (SH-p72, 5 µM) and (c)
after hybridization with target DNA (p-72, 1 µM) by the electrical
field.
electrodes were reacted with single stranded p72 (1 µM) by
applying the electric field in the hybridization buffer. After
hybridization, voltammetric experiments were carried out in
5 mM potassium ferricyanide in 100 mM potassium chloride
buffer at 100 mV/s. When the bare gold electrode was
reacted with 1 µM p72, the Ipa values were almost the same
with that of the bare gold electrodes.
In Figure 4(c), the voltammetric data showed that when
the ssDNA-electrode was reacted with 1 µM p72, the Ipa
value was decreased, and the change ratio of oxidation peak
current [(I3/I2−1) × 100, where I2 is oxidation peak after
immobilization and I3 is oxidation peak after hybridization]
was 72.9%. It is considered that the decreased value is
derived from potassium ferricyanide ion due to DNA
hybridization. Reproducibility was observed to be kept when
the same experiments were repeated 10 or more times.
Figure 5. Cyclic-voltammograms of 5 mM potassium ferricyanide in
100 mM potassium chloride at 100 mV/s using (a) bare gold electrode,
(b) after probe-modified electrode (SH-m72, 5 mM)) and (c) after hybridization with target DNA (p72, 1 µM).
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Bull. Korean Chem. Soc. 2005, Vol. 26, No. 3
Figure 6. CVs of 5 mM potassium ferricyanide in 100 mM
potassium chloride using (a) bare gold electrode (I1), (b) probemodified electrode (SH-R72, 5 µM; I2) and (c) after hybridization
with target DNA (p72, 1 µM; I3), where I1 is oxidation peak of bare
gold electrode, I2 is oxidation peak after immobilization and I3 is
oxidation peak after hybridization. (I2 − I1) × 100/I1 is −21.1% and
(I3 − I2) × 100/I2 is −21.5%.
These results suggest that target DNA, p72, can be detected
specifically by using potassium ferricyanide and ssDNAelectrode.
Electrochemical Gene Detection with the DNA Chip
Microarray. In Figure 5(a) and (b), the peak currents of
potassium ferricyanide decreased about 86.4% and the Ep
increased when the ssDNA-electrodes (SH-m72, 5 µM))
were used in comparison to those of the bare electrodes. The
ssDNAs (SH-m72) were used for non specific gene
detection. The ssDNA-electrodes were reacted with single
stranded p72 (1 µM)) with the same method as in Figure 4(c)
and measured the voltammetric experiments. In Figure 5(c),
after hybridization, the voltammetric data showed that when
the ssDNA-electrode was reacted with 1 µM p72, the Ipa
values were almost the same, and the change ratio of
oxidation peak current [(I3/I2−1) × 100] was 8.9%. However,
the change ratio of the peak current was −57.1%, when SHm72 reacted with m72 of the complementary DNA at the
same condition. It could be considered that the ssDNA
probes (SH-m72) almost did not hybridize with the targeted
gene.
In Figure 6(a) and (b), the peak currents decreased about
21.1% and the Ep increased when the ssDNA-electrodes
(SH-R72, 5 µM)) were used in comparison to those of the
bare electrodes. The ssDNAs (SH-R72) were used for nonspecific gene detection. The ssDNA-electrodes were reacted
with single stranded p72 with the same method as in Figure
4(c) and CV curves were measured. After hybridization, the
voltammetric data (Figure 6(c)) showed that when the
ssDNA-electrodes were reacted with 1 µM p72, the Ipa
values were almost the same, and decreased by 21.5%. It
could be considered that the ssDNA probes (SH-R72)
almost did not hybridize with the targeted gene.
Calibration Characteristics. Figure 7 shows calibrating
Yong-Sung Choi et al.
Figure 7. Calibration curves for concentration of target DNA p72
against (a) SH-p72, (b) SH-R72 and (c) SH-m72.
curves for concentration of the target DNA. In Figure 7(a),
the calibrating experiments for the target gene p72 against
SH-p72 probe DNA showed that the change ratio of peak
current values, [(I0/I − 1) × 100] where I is oxidation peak
after immobilization and I0 is oxidation peak after
hybridization, were linearly related to the concentration of
the target DNA p72 in the hybridization reactions ranging
from 10−18 to 10−5 M. In Figure 7(b) and (c), however, when
the target DNA p72 and the single base mismatched DNAs,
SH-m72 and SH-R72, were reacted, there were almost no
difference in the change ratio of peak current compared to
the complementary probe DNA ranging from 10−18 to 10−5
M. It is considered that the new indicator-free DNA chip
microarray can detect the targeted gene quantitatively with
high sensitivity.
Conclusions
In this study, the integrated microelectrode array was
fabricated on a slide glass using microfabrication technology. Probe DNAs consisting of thiol group at their 5’-end
were spotted on the gold microelectrode using micropipettes
utilizing the affinity between gold and the thiol group.
Cyclic-voltammogram of 5 mM ferricyanide in 100 mM
KCl solution at 100 mV/s confirmed the immobilization of
the probe DNA on the gold electrodes.
After hybridization of the target DNA using the electric
field, when several DNAs were detected electrochemically,
there was a difference between target DNA and mismatched
DNA in the anodic peak current values. It was derived from
ferricyanide ion due to hybridization of target DNA.
Immobilization of the probe DNA and hybridization of the
target DNA could be confirmed by fluorescent. This
indicator-free DNA chip microarray resulted in sequencespecific detection of the target DNA quantitatively ranging
from 10−18 M to 10−5 M in the buffer solution. These results
suggest that target DNA can be detected specifically by
using the indicator-free DNA chip array.
In principle, this method requires non-labeling of target
Hybridization by an Electrical Force and Electrochemical Genome Detection Bull. Korean Chem. Soc. 2005, Vol. 26, No. 3
DNA. This feature provides simple pretreatment of target
DNA. It suggested that multichannel electrochemical DNA
microarray is useful in developing a portable device for a
clinical gene diagnostic system. Advantages of this method
are simplicity in process and wide applicability. This method
can also be applicable to a new detection technology to
develop various biosensors.
Acknowledgement. This work was supported by grant
No. (R08-2003-000-10312-0) from the Basic Research
Program of the Korea Science & Engineering Foundation.
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